Second Messenger Systems

Second Messenger
Systems
Dr. Kunal A. Chitnis
2nd Yr Resident
T.N.M.C.
16th July 11

 Molecules that relay signals from receptors on the cell surface
to target molecules inside the cell i.e. cytoplasm or nucleus
 Relay the signals of hormones like epinephrine, growth factors
& others; causing some kind of change in the activity of the cell
 The term was coined upon the discovery of these substances
in order to distinguish them from hormones & other molecules
that function outside the cell as “first messengers” in the
transmission of biological information

 Earl Wilbur Sutherland Jr.
discovered second messengers
won the 1971 Nobel Prize in Medicine
 He saw that epinephrine would
stimulate the liver to convert glycogen
to glucose in liver cells, but
epinephrine alone would not convert
glycogen to glucose
 He found that epinephrine had to trigger a
second messenger, cyclic AMP for the liver to convert
glycogen to glucose

cAMP
System
cGMP
System
Phospho-
inositol
System
Tyrosine
Kinase
System
Ligands Epinephrine
Ach
ANP, NO Oxytocin PDGF
Primary
Effector
Adenyl
cyclase
Guanylate
cyclase
Phospho-
lipase C
Receptor
Tyrosine
kinase
Secondary
messenger
cAMP cGMP IP3 & DAG;
Ca2+
-

G- Proteins
 Guanine nucleotide binding proteins which act as a Transducer
between a receptor & an effector
 Discovered by Alfred Gilman & Martin Rodbell in 1990
 Significance:
• Of the top 100 drugs,
26 directed at GPCRs
• 60 % act through GPCRs
• 40 % of prescriptions
• 3rd largest family of genes (865)
• Present in almost every organ system

Endogenous Ligands:
Sensory signal mediators: Light & Olfactory stimulatory molecules
Biogenic amines: Dopamine, Adr, NA, Ach, Histamine
Peptide hormones: FSH, GnRH, calcitonin, TRH, Oxytocin
Mediators of inflammation: Prostaglandins, PAF, leukotrienes,
chemokines

Structure:
 Embedded in the plasma membrane
 7 transmembrane -helices
 Rhodopsin was first of these
to have its structure confirmed by
X-ray crystallography
-Adrenergic
Receptor
PDB 2RH1
Lysozyme
insert
ligand

Binding Domains
 Small molecular ligands
bind to sites within the
hydrophobic core formed by
transmembrane α helices
 Protein and peptide agonists
bind to N terminus &
extracellular hydrophilic
loops joining the
transmembrane domain
 G-proteins bind either to
second and third
cytoplasmic loop which is
largest or to carboxy terminus

 Molecular Switch: On/Off
 Heterotrimeric
α-subunit
β-subunit
γ-subunit
 α subunit: specific recognition of
receptors & effectors;
GTP binding site
 βƔ subunit: Membrane localisation by prenylation of Ɣ subunit
 Coded by of genes:
α – 23, β-7, Ɣ-12

Functions of α subunit & βƔ dimer

Receptors Couplers
Muscarinic Gi, Go, Gq
Dopamine D2 Gi, Go
β-adrenergic Gs, Gi
α2-adrenergic Gi, Gs, Go
GABA-B Gi, Go
5-HT Gi, Gq, Gs

G Protein Activation
 Conformational change
in receptor
 Transmitted from ligand
binding pocket to 2nd & 3rd
intracellular loop
 α subunit exchange GDP
with GTP
 Presence of GEF‟s
(Guanine exchange factors)
 Release of GTP bound
α subunit & βƔ dimer

Inactivation
 Activated α subunit is
inactivated by hydrolysis
of GTP to GDP by GAPs
(GTPase Activating
Proteins)
 Rebinds to βƔ complex
 Modulated by
Regulators of G
proteins Signaling (RGSs)
 Acclerate hydrolysis of
GTP & potential drug
targets

Toxins
 Cholera toxin catalyzes covalent modification of Gs
• ADP-ribose is transferred from NAD+ to an arginine residue
at the GTPase active site of Gs
• ADP-ribosylation prevents GTP hydrolysis by Gs
• The stimulatory G-protein is permanently activated.
 Pertussis toxin catalyzes ADP-ribosylation at a cysteine residue
of the inhibitory Gi, making it incapable of exchanging GDP for
GTP
• The inhibitory pathway is blocked.
 ADP-ribosylation is a general mechanism by which activity of
many proteins is regulated, in eukaryotes & prokaryotes.

Receptor Desensitization
 Activated receptor is
phosphorylated via a
G-protein Receptor Kinase
 Binds to a protein arrestin
promotes removal of the
receptor from the membrane by
clathrin-mediated endocytosis
 May also bind a cytosolic
Phosphodiesterase, bringing
this enzyme close to where
cAMP is being produced,
contributing to signal turnoff

Adenyl Cyclase
 9 membrane bound, 1 soluble isoform (AC1-10)
 120kDa
 Basal enzymatic activity modulated by
GTP liganded α subunit Gs & Gi
 Other regulatory interactions: βƔ subunits, Ca2+, protein
kinase & diterpene forskolin
 Dephophorylation of ATP by removal of 2 phosphate
molecules→ cAMP

cAMP has following major targets:
1. cAMP dependent Protein Kinase A (PKA)
2. cAMP regulated Guanine nucleotide exchange factors
termed EPACs (Exchange Proteins Activated by cAMP)
3. CREB (cAMP responsive element binding protein)

Protein Phosphorylation→ most common form of post-
translational modification in nature
Protein function altered by addition of a negatively charged
phosphate group to a Ser, Thr or Tyr residue by Protein Kinase:
• Binding properties
• Enzymatic activity if a catalytic protein
Protein phosphatase catalyzes removal of the Pi by hydrolysis
Protein OH + ATP Protein O P
O
O
O
+ ADP
Pi H2O
Protein Kinase
Protein Phosphatase

1. Protein Kinase A
 Holoenzyme: 2 Regulatory(R) & 2 Catalytic(C) subunits
 Heterotetramer complex R2C2
 ↓ cAMP, R inhibits C→ inactive
 ↑ cAMP→ 4 cAMP mols bind to R2C2, 2 to each R→ lowers
affinity to C → Activation

Active C subunits→
 Phosphorylate serine/threonine residues on proteins
As protein expression varies from cell type to cell type,
proteins that are available for phosphorylation will depend upon
the cell type
 Phosphorylation of ion channels→ Regulation
(Ca2+ activated K+ channel activation & Na+/K+ ATPase)
 Inhibition of Myosin Light Chain Kinase

Cell Type Stimulators Inhibitors Effects
Hepatocyte Epinephrine(β),
Glucagon
Produce glucose:
stimulate glycogenolysis,
inhibit glycogenesis,
stimulate gluconeogenesis,
inhibit glycolysis.
Skeletal -
Myocyte
Epinephrine(β) Produce glucose:
stimulate glycogenolysis,
inhibit glycogenesis,
stimulate glycolysis
Cardio-
myocyte
Norepinephrine
(β)
Sequester Ca2+ in
sarcoplasmic reticulum,
Phosphorylates
phospholamban
Actions of Protein kinase A

Smooth muscle
myocyte
β2 agonist (β2)
Histamine (H2)
Prostacyclin
Prostaglandin
D2/E2
Muscarinic
(M2)
Vasodilation
Adipocyte Epinephrine (β),
Glucagon
Enhance
lipolysis
Neurons in
Nucleus
accumens
Dopamine (D3) Activate
reward
system
Principal Cells Vasopressin
(V2)
Synthesis &
Exocytosis of
Aquaporin 2
Juxtaglomerular
Cells
Adrenergic (β1)
Dopamine(D1)
Glucagon
Renin
secretion

2. cAMP Response
Element-Binding
(CREB)
 Cellular transcription factor
 Genes: c-fos, the neurotrophin
BDNF, tyrosine hydroxylase &
many neuropeptides
 Neuronal plasticity & long-term
memory formation
 Development & progression of
Huntington‟s Chorea

3. Exchange Proteins Activated
by cAMP (EPAC)
 cAMP Regulated Guanine Nucleotide
Exchange Factors
 Bind to GDP liganded GTPase,
exchange of GDP for GTP
 Activation of PKC
 Cell differentiation/proliferation, cytoskeletal
organization, vesicular trafficking & nuclear transport
 Additional effector system
 Potential target for cancer therapy

Therapeutic Applications
Selected Drugs that target cAMP signalling
Drug MOA cAMP Therapeutic
Application
Salmeterol β2 Agonist ↑ Asthma, COPD
Rimonabant CB-1 Antagonist ↑ Obesity
Haloperidol D2,D3,D4 Antag ↑ Schizophrenia
Metoclopramide D2, 5HT4 Antag ↑ Nausea,
Vomiting
Desmopressin V2 Receptor
Agonist
↑ Diabetes
insipidus

Metoprolol β1 Antag ↓ Angina,
Hypertension,
CHF
Morphine μ Agonist ↓ Pain
Sumatriptan 5-HT1D/5-HT1B ↓ Migraine
Ibuprofen Non selective
Inhibitor of COX
↓ Inflammation,
Pain
Ranitidine H2 Antag ↓ Peptic ulcer,
GERD
Misoprostol PG Receptor
Agonist
↓ Prevention of
NSAID ulcers
Cabergoline D2 Agonist ↓ Parkinson‟s
disease

Novel Drug Targets:
 Analgesia
• ↑ cAMP→ ↑nociception
• AC1 & AC5 involved
• Selective AC1 & AC5inhibitors→ Analgesics
• Preclinical stages
 Drug dependence
• Repeated opiod exposure→ upregulation of AC activity
• 3 specific isoforms- AC1, AC5 & AC8
• Selective inhibitors for opiod dependence
 Neurodegenerative disorders
• Target second messengers used my multiple neurotransmitters
• AC1 is most attractive target
• AC1 activators→ used for cognitive decline

 Chronic Heart Failure:
• Alterations in β adrenoreceptor- AC- cAMP pathway
• Downregulation of β1 receptors
• Upregulation of inhibitory G proteins &
G-Protein coupled receptor kinases
• Catecholamine refractoriness, Exercise intolerance
• Protective phenomenon→ shields myocytes from
arrhythmogenic, hypertrophic & apoptotic effects of
catecholamines
• Genetic variant of G-Protein Coupled Receptor Kinase 5
(GRK5)→Accelerated desensitization→ Better prognosis
 Sperm function:
• sAC regulates sperm motility & capacitation
• ↑cAMP→ mediates capacitation
• Inhibitors for male contraception in early stages

 Unlike cAMP, cGMP has established signaling roles in only
few cell types
 Signaling pathways that regulate synthesis include:
1. Nitric Oxide
2. Hormonal Regulation (ANP/BNP/CNP)

1. Nitric Oxide
 Nitric oxide: diatomic free radical gas
 Lipid soluble
 Very small→ easy passage between cell membranes
 Short lived, degraded or reacted within a few seconds
 Synthesis:
Synthesized from L-arginine catalyzed by
reaction of NO-synthase
to form NO and L-citrulline

 Nitric Oxide Synthase (NOS) exists as 4 isoforms
 neuronal type I isoform (nNOS)
 inducible type II isoform (iNOS)
 endothelial type III isoform (eNOS)
 mitochondrial isoform (mtNOS)

NOS constitutively expressed in:
 eNOS :
• Endothelium, cardiac myocytes, renal mesangial cells,
osteoblasts, platelets
 nNOS :
• CNS and NANC nerves

Control exerted in following ways:
 Endothelium-dependent agonists (Ach, bradykinin, substance P)
↑ cytoplasmic concentration of Ca2+ ↑calcium-calmodulin 
eNOS or nNOS activation
 Shear stress in resistance vessels→ Sensed by
mechanoreceptors→ Transduced via a serine threonine protein kinase
called Protein kinase B/ Akt → Phosphorylation of specific residues on
eNOS

 iNOS:
• Macrophages, Kuffer cells, neutrophils, fibroblasts, vascular
smooth muscle cells & endothelium
• Activity independent of Ca2+
• Positive inducers : IFNγ, TNFα, IL-1, IL-2, LPS, antigens
• Inhibitory cytokines: TGF-β, IL-4, IL-10, glucocorticoids

2. Natriuretic Peptide Receptors
3 small peptide ligands:
 Atrial Natriuretic Peptide (ANP):
• Released from atrial storage granules
• ↑ Intravascular volume/ Stimulation with pressor hormones
 Brain Natriuretic Peptide (BNP):
• Synthesized and released from ventricular tissue
• Volume overload

 C-type Natriuretic Peptide (CNP):
• Synthesized in brain & endothelial cells
• Growth factors/ stress on vascular endothelial cells
 Major physiological effects:
 ↓ BP (ANP/BNP)
 ↓ Cardiac hypertrophy & fibrosis (BNP)

Receptors with intrinsic enzymatic activity
3 types of receptors:
1) ANP Receptor (NPR-A)
 Binds to ANP & BNP
 Maintaining normal state of CVS
2) BNP Receptor (NPR-B)
 Binds to BNP
 Role in bone function
3) CNP Receptor (NPR-C)
 No enzymatic activity

 Ligand brings juxtamembrane regions together
 Phosphorylation of serine residues→
Stimulation of guanyl cyclase

Guanylate Cyclase:
 Membrane-bound (type 1) & soluble (type 2) forms
 Membrane bound→ activated by ligands
 Soluble→ activated by NO
 Catalyzes reaction of
GTP to cGMP

Downstream reactions of cGMP:
 Activation of Protein Kinase G
 cGMP gated ion channels
 cGMP-modulated Phosphodiesterase

cGMP-dependent protein kinase or Protein Kinase G
(PKG)
 Serine/threonine kinases
 PKG I → cytoplasm, PKG II → membrane associated
 PKG I→ smooth muscles, platelets, brain
PKG II→ intestines, bones, kidney
 Regulatory and catalytic domains contained on a single
polypeptide chain
 Upon activation dimerizes to form PKG holoenzyme

 Actions of PKG I:
 Inhibits of Gq/G11→ Inhibition of Phospholipase C→ ↓ Ca2+
 Inhibits G12/G13→ Inhibits Ca2+ sensitizing mechanisms
 Phosphorylates and inhibits the Myosin light chain
kinase which normally phosphorylates the myosin light
chains

 Effects:
 Relaxes all smooth muscle types
 Inhibits platelet aggregation & granule secretion
 Functions of PKG II:
 Stimulates in chloride & water secretion in small intestine
 Normal endochondral bone development
 Inhibits renin secretion

1. NO Donors
 Organic nitrates
• Short acting: Nitroglycerin (NTG)
• Long acting: Isosorbide mononitrate, Isosorbide dinitrate,
Pentaerythritol tetranitrate
• Use: Treatment of Angina
• A/E: Tolerance (Nitrate free interval)

 Sodium Nitroprusside
• Hypertensive emergencies
 Nicorandil
• K+ channel opner and NO donor
• Antianginal
 CCB’s: Nitrendipine, Nifendipine, Lacidipine
• Dihydropyridine
• Release NO
• Retard atherosclerosis
• Use : Hypertension, angina

 β Blockers: Nebivolol, Celiprolol
• Additional vasodilatory effects
2. Natriuretic Peptides
 Nesiritide:
• Synthetic Brain Natriuretic Peptide
• Promotes vasodilation, natriuresis & diuresis
• Use: Acutely decompensated congestive heart failure
 Ecadotril:
• Neural Endopeptidase (NEP) catalyses BNP degradation
• It inhibits NEP
• Use: CHF (Phase 2)

BAY series of compounds
8-bromo-cGMP
8-bromoPET-cGMP
8-pCPT-cGMP
 Specific activators & inhibitors of cGKI & cGKII
 Improve the specificity & availability of treatment
 Evaluated for:
• Asthma
• Graft survival after liver & lung transplantation

Phosphodiesterases (PDE)
 PDE comprise family of enzymes expressed in almost all cells
of the body & of prime importance in cellular functioning
 Hydrolysis of phosphodiester bond in the second messengers
c AMP & c GMP → inactive forms 5 AMP and 5 GMP
respectively

 Reversal of activation of cellular protein kinases
 Inactivation leads to termination of intracellular signals
 Regulators of cyclic nucleotide signaling & responsible for
diverse physiological functions
 PDEs comprise a super family composed of 25 genes
Categorized into 11 sub families i.e. PDE 1 – PDE 11
 Substrate specificity:
c AMP
Specific
c GMP
Specific
cAMP & cGMP
Specific
• PDE 4
• PDE 7
• PDE 8
• PDE 5
• PDE 6
• PDE 9
• PDE 1
• PDE 2
• PDE 3
• PDE 10
• PDE 11

 Act by modulating the levels of 2nd messengers
Therapeutic Applications:
 Congestive heart failure:
 PDE-3 Inhibitors→ Amrinone, Milrinone
 ↑ In force of contraction
 Direct vasodilation of both the resistance & capacitance vessels
 Inodilators

 Erectile dysfunction:
 PDE-5 Inhibitors→ Sildenafil, Tadalafil, Vardenafil
 Never combined with nitrates:
• Nitrates produce vasodilatation by NO dependent elevation
of cGMP in vascular smooth muscle
• Thus PDE 5 inhibitor if given along with of a NO donors can
cause profound & extreme hypotension
• Pts should be asked for a history of nitrate consumption within
previous 24 hrs

 Bronchial Asthma:
 Theophylline, Aminophylline
 Non selective PDE inhibitor
 Narrow therapeutic range (5 – 20 mcg/ml)
Other uses: COPD, Premature apnoea in infants
 Peripheral Vascular Disease:
 Pentoxyphylline
 PDE 3 inhibitor
 Rheologic modifier improves the flexibility of RBCs
& ↓ blood viscosity
 Improves microcirculation
 Cilastazol
 PDE 3 inhibitor
 Promotes vasodilatation & inhibition of platelet aggregation

 Antiplatelets
 Dipyridamole
 Inhibits PDE 5
 Prevents uptake & degranulation of adenosine
 Was introduced for angina pectoris but was a failure due to
„coronary steal phenomenon‟
 Anagrelide
 PDE 3 inhibitor
 Other uses: Essential thrombocytosis,
 Thrombocytopenia in Polycythmia Vera
 Zaprinast
 PDE 6 Inhibitor
 Antiproliferative & proapoptic property
 Vasoproliferative disorders

 Antispasmodics
 Drotaverine
 Inhibits PDE 4
 Selective for smooth muscles
 Intestinal, biliary, renal colic; Irritable bowel syndrome;
Dysmenorrhea; Acceleration of labor

Phospholipase C: IP3-DAG Pathway

Three Types of Inositol phospholipids:
PI, PI(4)P, PI(4,5)P2

Phospholipase C exists as 2 isoforms:
1. Phospholipase Cβ:
• Activated by GPCRs couple to Gi/Gq→ release GTP bound
α subunit & βƔ dimer→ both activate
2. Phospholipase CƔ:
• Activated by Receptor/Non Receptor Tyrosine Kinases
 Cytosolic enzymes
 Translocate to plasma membrane on receptor activation
 Ligands:
AGT, GnRH, GHRH, Oxytocin, TRH

Phospholipase C forms Diacylglycerol & Inositol 1,3,5- triphosphate
from Phosphotidyl Inositol 4,5-bisphosphate

Protein kinase C:
 Regulatory domain & catalytic
domain tethered together by a
hinge region
 C1 domain, present in all of the
isoforms of PKC has a binding site
for DAG
 C2 domain acts as a Ca2+ sensor
 Catalytic Region brings about
phosphorylation Ser/Thr a.a. of
proteins
 Upon activation, translocated to the
plasma membrane

Cell type Activators Effects
Smooth muscle
(vascular)
5HT(5HT2A)
Adrenergic(α1)
Vasoconstriction
Smooth muscle
(GIT)
5HT(5HT2A/5HT2B)
Adrenergic(α1)
Contraction
Smooth muscle
(bronchi)
5HT(5HT2A)
Adrenergic(α1)
Ach(M1/M3)
Bronchoconstriction
Smooth muscle
(ureter/ urinary bladder/
urethral sphincter)
Adrenergic(α1) Contraction
Smooth muscle
Iris dilator Adrenergic(α1) Contraction
Iris constrictor/ Ciliary Ach(M3) Constriction
Platelets 5HT(5HT2A) Aggregation

Cell type Activators Effects
Cardiomyocytes Adrenergic(α1) Positive ionotropic
effect
Hepatocyte Adrenergic(α1) Glycogenolysis,
Gluconeogenesis
Adipocyte Adrenergic(α1) Glycogenolysis,
Gluconeogenesis
Proximal
Convoluted tubule
Angiotensin II (AT1)
Adrenergic (α1)
Stimulate NHE3→
H+ secretion & Na+
reabsorption
Stimulate
basolateral Na+-K+
ATPase →
Na+ reabsorption

 IP3 Receptor
 Ligand gated Ca2+ channel
 High conc. in membrane of ER
 Ligands which regulate:
1. PKA:
↑ Ca2+ release by phosphorylation
2. PKG:
• Inhibits Ca2+ release
3. IP3:
• ↑ Ca2+ release
4. Ca2+:
• Conc of 100-300nM→ ↑ Ca2+ release
• Conc of 1000nM→inhibits Ca2+ release
• Oscillatory pattern of Ca2+ release

 Ryanodine receptor
 Present in skeltetal & cardiac muscles
& neurons
 Major cellular mediator of
calcium-induced calcium release
 Ca2+ enters through L-type Ca Channels
 Conformational change in RyR receptors
 Release of Ca2+ from SR into cytosol
 Agonist: Xanthines (Caffeine, Pentoxyfylline)
 Antagonist: Dantrolene

 Calcium Reuptake
 Na+/Ca2+ exchanger on plasma membrane
 Ca2+ pump on ER membrane
 Ca2+ binding molecules
 Ca2+ pump on Mitochondia

Cell Type Effect
Secretory Cells (mostly) ↑secretion (vesicle fusion)
Juxtaglomerular cells ↓secretion
Parathyroid chief cells ↓secretion
Neurons
transmission (vesicle
fusion)
T-cells
activation in response to
antigen presentation
Myocytes Contraction (TroponinC)
Effects of Ca2+ :

Calmodulin binds Ca2+ & activates 5 different
Calmodulin-dependent kinases
1. Myosin light-chain kinase→ Phosphorylates myosin→
Contraction in smooth muscle
2. CaMK I → synaptic function
3. CaMK II → neurotransmitter secretion, transcription factor
regulation & glycogen metabolism
4. CaMK III → protein synthesis
5. Calcineurin, a phosphatase that inactivates Ca2+ channels
by dephosphorylating prominent role in activating T cells →
inhibited by some immunosuppressants

Therapeutic Applications:
Drug Mechanism of
Action
IP3/DAG
Levels
Therapeutic
Application
Prazosin α1 blocker ↓ Hypertension/
Prostate
Hyperplasia
Chlorpheni-
ramine
H1 blocker ↓ Allergies/
Common cold
Ipratropium
bromide
M3 blocker ↓ Asthma/ COPD
Losartan AT1 Receptor
blocker
↓ Hypertension/
MI/ Diabetic
Nephropathy
Montelukast LT C4/D4 blocker ↓ Asthma
Oxytocin Direct action on
Gq
↑ Labour induction/
Uterine inertia

Cell surface receptors recruit activity of protein kinases in two general ways:
Receptor Tyrosine Kinases:
Possess an intrinsic tyrosine kinase activity that is part of the receptor protein
Examples include receptors for growth factors (PDGF, EGF, insulin, etc.)
Non-receptor tyrosine kinases:
Receptors lacking self-contained kinase function
recruit activities of intracellular protein kinases to the plasma membrane

Receptor Tyrosine Kinases
 Implicated in diverse cellular responses:
Cell division, Differentiation & Motility
 At least 50 RTKs identified:
Subdivided into 10 subclasses based on
differences within extracellular, ligand-binding domain of receptor

Structure:
Four common structural features shared
among RTKs
 Extracellular ligand-binding domain
 Single transmembrane domain
 Cytoplasmic tyrosine kinase domain(s)
 Regulatory domains

Receptor Dimerisation
Three ways in which signaling proteins can cross-link receptor
chains
1. Dimer ligand
2. Monomer but brought together by proteoglycan
3. Cluster on membrane

 Receptor dimerization leads to activation of
catalytic domains causing
autotransphosphorylation
 Receptor autotransphosphorylation:
• Further stimulates kinase activity
• Leads to phosphorylation of additional
proteins involved in receptor
signalling pathway

Provides “docking sites” for downstream
signalling proteins
(Grb2, PI3-kinase, phospholipase C , etc.)
Src homology (SH) 2 & SH3 domains:
SH2 domains: bind P-Tyr-containing sequences
SH3 domains: bind to pro-rich (PxxP) sequences
Activates Phospholipase CƔ
The binding of SH2-containing intracellular signaling
proteins to an activated PDGF receptor

RTK mediated pathways:
1. Ras-Raf-MAP kinase pathway
The activation of Ras by RTK signaling

The MAP-kinase regulated by Ras

Ras-Raf-MAP kinase pathway in Cancer
 Ras gene mutation→
defective Ras protein
 GAP binds to GTP bound Ras, but
not able to provide domain for GTPase
 GTP is not lysed & the GTP bound
Ras remains continuously active
 Thus permanently activated MAP kinase pathway
results in growth factors transcription causing continuous cell
proliferation
 Development of Cancer

2. PI3 Kinase Pathway
 Activated PI3 docks at the
phosphorylated RTK
 Brings about phosphorylation of
PIP2→PIP3
 Downstream effects:
• Inhibits proapoptotic protein BAX
• Translation of tumour proteins by
activation of mTOR
• Phosphorylation of FOXFO, antitumour protein,
→ ubiquitinisation→ degradation

I. Receptor Tyrosine Kinase Inhibitors:
A. Epidermal Growth Factor Receptor Inhibitor/ HER 1 Inhibitor
Geftinib:
• Inhibits EGFR tyrosine kinase activity
• Blocks ATP binding site
• NSCLC pts. who have failed with std. chemotherapy
 Erlotinib
• Similar mechanism of action
• Locally advanced or metastatic NSCL & Pancreatic Ca

 Cetuximab:
• Monoclonal antibody to extracellular domain of EGFR
• Combined with Radiation for Locally advanced
Squamous Cell Ca of head & neck
• EGFR positive metastatic Colorectal Ca
 Panitumumab:
• Recombinant fully humanized IgG to extracellular domain
of EGFR
• EGFR positive metastatic Colorectal Ca

B. HER2/neu Inhibitor
 Lapatinib
• Inhibits EGFR & HER2/neu Kinase activity
• ATP binding pocket
• Approved for Trastuzumab Refractory breast Ca with
Capecitabine
 Trastuzumab:
• Humanized monoclonal Antibody to external domain
of HER2/neu receptor
• Her2/neu overexpressing metastatic breast Ca
with Paclitaxel

II. mTOR inhibitors:
 IL-2 stimulates immune system by activation of T Cells via
activation of mTOR
Sirolimus (Rapamycin)
 Binds to mTOR & inhibits action of IL-2
 Immunosuppressive agent in organ transplant & GVHD
 Cardiac stents to ↓ chances of re-occlusion
 A/E: thrombocytopenia, hyperlipidemia, HUS
Everolimus
 Short t1/2
 Cardiac transplants

 JAK-STAT Receptor Pathway
 Ligands: Interferon Ɣ, Growth hormone, Prolactin
 Receptors have no intrinsic activity
 Intracellular domain binds intracellular tyrosine kinase→
Janus Kinase (JAK)
 Receptor mediated dimerization → phosphorylation of
Signal transducers & activators of transcription (STAT)
 STATs translocate to nucleus & regulate transcription
 4 JAKs & 6 STATs combine differently depending on
Cell type & signal
 Eg: Prolactin→ JAK1, JAK2 & STAT5

Therapeutic Application:
 Lestaurtinib:
• Janus Kinase 2 Inhibitor
• JAK/STAT signaling exaggerated in MPNs
• Polycythemia vera, essential thrombocythemia
& primary myelofibrosis
• Mutant JAK2 activity
• Inhibits wild type JAK2 kinase activity
• Inhibits proliferation MPD cells
• Phase II AML & Myeloproliferative disorders

 Receptor Serine-Threonine Kinases:
 Anologous to RTK except they have
Serine/Threonine kinase domain in cytoplasmic region
 Ligand: TGFβ
 Dimerizes in presence of ligand→
Phosphorylation of kinase domain→ activation
 Phosphorylation of gene regulatory protein termed
Smad on serine residue
 Dissociates from receptor→ associates with transcription factors
→ Morphogenesis & transformation
 Inhibitory Smads: Smad6/7

 Toll like Receptors
 Signaling related to innate immunity
 Family of 10 receptors
 Structure:
• Single polypeptide chain
• Large extracellular ligand binding domain
• Short membrane spanning domain
• Cytoplasmic TIR domain
 Ligands: Pathogens (lipids, peptidoglycans, lipopeptides,
viruses)
 Inflammatory response to pathogen

Signaling:
 Receptor induced dimerisation
 Recruitment of Mal & MyD88 to TIR
 Recruits Interleukin-associated kinases
(IRAKs)
 Auotphosphorylates & complex with MyD88
 Also recruits TRAF6
 Interact with protein kinase TAK1 & Adaptor TAB1
 Activates NF-КB
 Trasncription of inflammatory genes

TNFα Receptors
 Structure:
• Single membrane spanning receptor
• Extracelluler ligand binding domain
• Transmembrane domain
• Cytoplasmic domain (death domain)
 2 types: TNF1 (most cells) & TNF 2 (immune cells)
 Activated by trimerisation

Has following effects:
 Activation of NF-κB:
• Transcription of proteins involved in cell survival and
proliferation, inflammatory response & anti-apoptotic factors
 Activation of the MAPK pathways:
• The JNK pathway is involved in cell differentiation,
proliferation & is pro-apoptotic
 Induction of death signalling:
• Cell apoptosis

Therapeutic Application
 Promotes inflammatory response & associated with
autoimmune disorders:
(Rheumatoid arthritis, ankylosing spondylitis, inflammatory
bowel disease, psoriasis, hidradenitis suppurativa and
refractory asthma)
 Treated by using a TNF inhibitor:
• Monoclonal antibodies such as
Infliximab, Adalimumab & certolizumab pegol
• Circulating receptor fusion protein such as etanercept

Pharmacodynamic Interactions in a Multicellular
Context
 Consider the vascular wall of an arteriole
 Several cells interact at this site:
Smooth muscle cells(SMC), endotheial cells, platelets & post-
ganglionic parasympathetic neurons
 Effects produced
• SMC contraction→ Ang II, NE
• SMC relaxation→ NO, BNP, epinephrine
• Alter gene expression→ PDGF, Ang II, NE, Eicosanoids

 Patient with hypertension
• ↑ levels of AngII
• ↑ activity of sympathetic nervous system
• ↓ NO production
 Pharmacotherapy directed towards:
• ↓ BP
• Prevent long term changes in vessel wall
 Drugs used to treat hypertension
• ↓ Ang II (Atenolol/Aliskiren/Enalapril/Losartan)
• α1 blockers→ ↓ NE binding on SMCs
• ↑ NO production (Na Nitroprusside)

Conclusion:
 Second messengers have shown to play physiological &
pathophysiological settings
 Evolved as targets for drug develoment for numerous diseases
However, availability of compounds acting on specific targets is
the biggest challenge
 Also is their difficult expression & purification
 Such specific drugs are still in their early stages of development
„Today‟s drug targets, tomorrow‟s blockbusters‟

Second Messenger Systems

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